ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane

Joyce, Charles; Shelness, Gregory S.; Davis, Matthew; Lee, Richard G.; Skinner, Kelly; Anderson, Richard A.; Rudel, Lawrence L.

doi:10.1091/mbc.11.11.3675

Cited by 106 publications

(129 citation statements)

References 36 publications

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“…ACAT2 has only two transmembrane domains, with both the N and C termini facing the cytosol (29). A second study reported that both ACAT1 and ACAT2 have five transmembrane domains, with their N and C termini on opposite sides of the ER membrane (28). It has been speculated that the different membrane topologies observed for ACAT1 and ACAT2 may be due to significant differences in the amino acid sequences within the putative transmembrane domains (29).…”

Section: Discussionmentioning

confidence: 99%

“…The membrane topologies of human ACAT1 and ACAT2, which share 15-25% identity with DGAT1, have been determined. ACAT1 appears to have 5-9 transmembrane domains with the N terminus facing the cytosol and the C terminus in the ER lumen (27,28). Two different models have been proposed for human ACAT2.…”

mentioning

confidence: 99%

“…One model proposes that ACAT2 has two transmembrane domains, and both the N and C termini face the cytosol (29). The second model suggests that ACAT2 has five transmembrane domains, and the termini are on opposite sides of the ER membrane (28).…”

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Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

McFie

Stone

Banman

et al. 2010

Journal of Biological Chemistry

130

125

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Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols. Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of synthesizing diacylglycerol, retinyl, and wax esters in addition to triacylglycerol. Here, we report the membrane topology for murine DGAT1 using protease protections assays and indirect immunofluorescence in conjunction with selective permeabilization of cellular membranes. Topology models based on prediction algorithms suggested that DGAT1 had eight transmembrane domains. In contrast, our data indicate that DGAT1 has three transmembrane domains with the N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which accounts for ϳ50% of the protein, is present in the endoplasmic reticulum lumen and contains a highly conserved histidine residue (His-426) that may be part of the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1 to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is not required for the catalytic activity of DGAT1 but, instead, may be involved in regulating enzyme activity and dimer/tetramer formation. Acyl coenzyme A:1,2-diacylglycerol acyltransferase (DGAT)2 is a membrane-bound enzyme that catalyzes the biosynthesis of triacylglycerols (TGs) (1). TGs are a class of neutral lipid that represents the major form of stored energy in eukaryotic organisms (2). However, excessive accumulation of TG in tissues can lead to obesity and insulin resistance, whereas increased TG in the blood is a risk factor for atherosclerosis (3)(4)(5).DGAT catalyzes the formation of an ester bond between a long chain fatty acid (fatty acyl coenzyme A (CoA)) and the free hydroxyl group of diacylglycerol (DG), generating TG. Two DGAT genes have now been identified, Dgat1 and Dgat2, which share no sequence homology. DGAT1 belongs to a large family of membrane-bound O-acyltransferases (MBOAT) that includes acyl-CoA:cholesterol acyltransferase-1 and -2 (ACAT-1 and -2), which catalyze cholesterol ester biosynthesis (6 -10). DGAT2 belongs to the DGAT2/acyl-CoA:monoacylglycerol acyltransferase gene family including monoacylglycerol acylCoA acyltransferases 1-3 and wax synthase (9, 11-16). Biochemical analyses have indicated that DGAT1 and DGAT2 have distinct roles in TG metabolism. When overexpressed in cells, DGAT2 yielded a much larger increase in intracellular triacylglycerol than DGAT1 (17). In in vitro assays, DGAT1, but not DGAT2, was capable of using a broad array of acyl acceptors to synthesize diacylglycerol, retinyl, and wax esters in addition to triacylglycerol (18).In vivo experiments in mice have also provided strong evidence that DGAT1 and DGAT2 do not serve redundant roles in lipid metabolism. Dgat1-deficient (Dgat1 Ϫ/Ϫ ) mice were viable with modest reductions in TG con...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

McFie

Stone

Banman

et al. 2010

Journal of Biological Chemistry

130

125

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“…Although the enzymological properties of ACAT isozymes are also very similar [41], the exact membrane topology and the active site of ACAT isozymes remain unresolved. Joyce et al reported that the active site of ACAT1 (Ser 269 ) is located in the cytoplasm, while that of ACAT2 (Ser 249 ) is located in the lumen [42]. On the other hand, Chang et al reported that the active site of ACAT1 (His 460 ) and ACAT2 (His 434 ) is located in the transmembrane domain [43,44].…”

Section: Discussionmentioning

confidence: 99%

Selectivity of Microbial Acyl-CoA : cholesterol Acyltransferase Inhibitors toward Isozymes

et al. 2007

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The selectivity of microbial inhibitors of acylCoA : cholesterol acyltransferase (ACAT) toward the two isozymes, ACAT1 and ACAT2, was assessed in cell-based assays. Purpactin A (IC 50 values of ACAT1 vs. IC 50 values of ACAT2; 2.5 mM vs. 1.5 mM), terpendole C (10 mM vs. 2.0 mM), C (32 mM vs. 0.36 mM) and D (38 mM vs. 1.5 mM) showed selective inhibition against ACAT2. In particular, pyripyropene A was found to be the most selective ACAT2 inhibitor with a selective index of more than 1,000.Keywords acyl-CoA : cholesterol acyltransferase, isozyme, microbial inhibitors, lipid droplet accumulation, pyripyropene, beauveriolide, atherosclerosis Introduction Acyl-CoA : cholesterol acyltransferase (ACAT), an ER membrane protein, is responsible for many functions in the body. ACAT has been recognized as a target for inhibition by a new type of antiatherosclerotic agents [1]. Many pharmaceutical laboratories have developed synthetic ACAT inhibitors. However, almost none of them could be successfully developed because of side effects or low in vivo efficacy [2]. Recent molecular biological studies revealed the existence in mammals of two different ACAT isozymes, ACAT1 and ACAT2 [3ϳ6]. ACAT1 is ubiquitously expressed in tissues and cells, while ACAT2 is expressed predominantly in the liver (hepatocytes) and intestine [7]. Therefore, it is important to determine the selectivity of inhibitors toward the two ACAT isozymes for their development as new antiatherosclerotic agents [8]. However, such data have rarely been reported so far [9,10].Our research group discovered a number of microbial ACAT inhibitors with an enzyme assay using rat liver microsomes; these inhibitors included pyripyropenes [11ϳ13], purpactins [14,15], glisoprenins [16ϳ18] and terpendoles [19ϳ21] (Fig. 1). The activities of these inhibitors in cell-based assays and their selectivity toward the two ACAT isozymes have not been fully studied. We also developed a cell-based assay of lipid droplet accumulation in mouse macrophages, and discovered beauveriolides [22ϳ25], phenochalasins [26,27], spylidone [28], sespendole [29] and K97-0239s [30] with this assay (Fig. 2). We studied the molecular targets of beauveriolides I and III in macrophages and found that they inhibited cholesteryl ester (CE) synthesis by blocking ACAT activity in macrophages, leading to the inhibition of lipid droplet accumulation. More importantly, they proved orally active in atherogenic mouse models and are expected to provide [31,32]. However, the molecular targets of the other inhibitors are not yet well defined, and their effect on ACAT activity as a potential target should be studied.Thus, we studied microbial inhibitors discovered by screening against ACAT ( Fig. 1) and lipid droplet accumulation ( Fig. 2) by examining their inhibitory activity against ACAT1 and ACAT2 isozymes, and their selectivity toward the isozymes. For the examination, two cell lines, CHO cells expressing African Green monkey ACAT1 (ACAT1-CHO) and ACAT2 (ACAT2-CHO) were used [9]. was purchased from Per...

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“…Like DGATs, two genes that encode ACAT1 and ACAT2 have been identified with distinct roles (35)(36)(37). Although the membrane topology of ACAT2 is still controversial (38,39), it secretes more VLDL cholesterol compared with ACAT1 when overexpressed in RH7777 cells (27). These data suggest that DGAT1 and ACAT2 might coordinately participate in VLDL formation within the ER lumen.…”

Section: Discussionmentioning

confidence: 99%

Increased Very Low Density Lipoprotein Secretion and Gonadal Fat Mass in Mice Overexpressing Liver DGAT1

Yamazaki

Sasaki

Kakinuma

et al. 2005

Journal of Biological Chemistry

130

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Acyl-CoA:diacylglycerol acyltransferases (DGATs) catalyze the last step in triglyceride (TG) synthesis. The genes for two DGAT enzymes, DGAT1 and DGAT2, have been identified. To examine the roles of liver DGAT1 and DGAT2 in TG synthesis and very low density lipoprotein (VLDL) secretion, liver DGAT1-and DGAT2-overexpressing mice were created by adenovirus-mediated gene transfection. DGAT1-overexpressing mice had markedly increased DGAT activity in the presence of the permeabilizing agent alamethicin. This suggests that DGAT1 possesses latent DGAT activity on the lumen of the endoplasmic reticulum. DGAT1-overexpressing mice showed increased VLDL secretion, resulting in increased gonadal (epididymal or parametrial) fat mass but not subcutaneous fat mass. The VLDL-mediated increase in gonadal fat mass might be due to the 4-fold greater expression of the VLDL receptor protein in gonadal fat than in subcutaneous fat. DGAT2-overexpressing mice had increased liver TG content, but VLDL secretion was not affected. These results indicate that DGAT1 but not DGAT2 has a role in VLDL synthesis and that increased plasma VLDL concentrations may promote obesity, whereas increased DGAT2 activity has a role in steatosis.Triglyceride (TG) 1 is the major energy storage form and is synthesized primarily in three tissues: liver, adipose, and small intestine. In the liver, synthesized TG is either stored in cytoplasmic droplets or secreted as very low density lipoprotein (VLDL) particles. Acyl-CoA:diacylglycerol acyltransferase (DGAT) is a membrane-bound enzyme that catalyzes the last step in the synthesis of TG. Classified by detergent sensitivity, two types of DGATs in microsomes have been proposed: the overt type (on the cytosol) catalyzes the synthesis of TG destined for cytoplasmic droplets, and latent type (on the lumen of the endoplasmic reticulum) catalyzes the synthesis of TG for VLDL formation (1). Because it has been well established that cytosolic droplet TG cannot be incorporated en bloc into VLDL (2), the relative activities of these two functions of DGAT may have a significant impact on the level of triglyceridemia as well as on the development of steatosis.Regarding the molecular aspects of DGAT, the cDNAs of DGATs, viz. DGAT1 and DGAT2, have been recently cloned and sequenced (3, 4). DGAT1 and DGAT2 are unrelated proteins that exhibit DGAT activity. DGAT1 is expressed ubiquitously, with the highest expression levels in the small intestine (3), and the phenotypes of DGAT1-null mice have been extensively examined (5-9). DGAT1-null mice are viable, can still synthesize TG, and have normal 4-h fasted plasma TG levels (5). These mice have reduced adiposity and are resistant to diet-induced obesity through a mechanism that involves increased energy expenditure (5). The increased energy expenditure is due partly to the increased peripheral leptin sensitivity in DGAT1-deficient mice (6). DGAT2 is expressed ubiquitously, with high expression levels in the liver and white adipose tissue (WAT) (4). In contrast to DGAT1-deficient ...

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ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane

Cited by 106 publications

References 36 publications

Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

Selectivity of Microbial Acyl-CoA : cholesterol Acyltransferase Inhibitors toward Isozymes

Increased Very Low Density Lipoprotein Secretion and Gonadal Fat Mass in Mice Overexpressing Liver DGAT1

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